Cullins are a recently identified family of evolutionarily conserved proteins linked to the ubiquitination of a potentially large number of cellular proteins. Characterization of a small number of cullins has identified critical functions in such diverse physiological processes as cell cycle and cell growth control, tumor suppression and animal development. We and others previously discovered a cullin-interacting RING finger protein, ROC1 (also known as Rbx1 or Hrt1), that carries essential function for CUL1-mediated SCF E3 ligases and for cell cycle progression. We have found that ROC1 and its homologue, ROC2, allosterically activates E2 and interacts with all cullin members to constitute many distinct ROC-cullin ubiquitin ligases in vivo. We have recently identified novel interactions between ROC-cullin ligases and two evolutionarily conserved proteins, p 120 TIP120 and p 127 DDB1. Our preliminary studies have led to the hypothesis that DDB 1 may function as a ROC-dependent E3 NEDD8 ligase toward cullin proteins, and that TIP120 proteins may function as assembly factors of various ROC-cullin ligases. We propose an investigation that combines biochemical, proteomic and genetic approaches in mammalian and fission yeast cells to determine the mechanisms and functions of TIP120 and DDB 1 and a poorly characterized CUL4. Through these concerted efforts, we anticipate a great opportunity to better understand the mechanism regulating the ROC-cullin family of ubiquitin ligases and the mechanism underlying the substrate recognition by this family of E3 ligases. Aim I. The function and mechanism of TIP120 1. Regulation of TIP120-cullin association by NEDD8 pathway in vivo2. Genetic analysis of fission yeast TIP120 3. Determination of the in vivo function of TIP120 in mammalian cells 4. Purification and analysis of TIP120 complexes.Aim II. Function of DDB1 as an NEDD8 E3 ligase.1. Biochemical reconstitution of NEDD8 ligation2. Genetic analysis of DDB 1 in fission yeastAim III. Function and complexes of CUL4A1. Determination of the function of human CUL4A2. Functional analysis of CUL4 in fission yeast3. Purification and characterization of CUL4 complexes.